It is well known that spheres may be prepared very readily, for example from sodium alginate, by using the property of alginate solutions to gel in the presence of cations such as, for example, calcium ions. The material to be encapsulated in the spheres is first dispersed in the aqueous alginate solution. This solution is added dropwise to an aqueous solution of a calcium salt. There is immediate gelation, which produces spheres of gelled alginate. The surface of the spheres may then be stabilized by immersion in a solution of a polycationic polymer such as poly-L-lysine or polyethyleneimine (Lim, F., U.S. Pat. No. 4,352,883, 1982). A membrane forms at the periphery, resulting from ionic association between the alginate and the polycation. This membrane allows small molecules to pass through while retaining large molecules and cells. It is then possible to liquefy the inner gel by immersing the alginate spheres, stabilized by the polycationic polymer, in a citrate solution, so as to chelate the calcium in the spheres (Lim, F., U.S. Pat. No. 4,352,883, 1982). The material incorporated then remains contained within the membrane.
This process has the great advantage of being carried out entirely in aqueous medium and of retaining excellent viability with respect to encapsulated living cells, which may multiply within the capsule. Hence, it is widely used for the inclusion of living tissues, cells and microorganisms. Thus, alginate spheres containing microorganisms are used in the food industry in order to carry out fermentations (fermentation of dairy products, beer, champagne, etc.). These techniques are applied to plant or animal cells or organs with the aim of cryoprotecting or producing metabolites, to animal cells or tissues (islets of Langerhans, hepatocytes, etc.) for implantation in human or veterinary medicine (cell therapy), or for carrying out toxicology tests in vitro. Cells are also cultured in such spheres for the production of biological substances such as the monoclonal antibodies developed by hybridomas, which accumulate in the spheres and are thus easily harvested after opening the membranes.
However, the process has drawbacks associated with the nature of the membrane. As it involves no covalent bonds but only ionic bonds between the alginate and a polycation, it is of limited stability (Dupuy et al., J. Biomed. Mat. Res., 1988, 22, 1061-1070). The polymers have a tendency to pass into solution over time. Moreover, if the pressure increases within the capsule under the effect of cell multiplication, the membrane cannot resist the pressure and cells are released into the medium. It is thus often necessary to apply successively alternate layers of alginate and then of polycation ("sandwiches") in order to obtain a solid membrane which does not allow the contents to diffuse out (Wong H. and Chang T. M. S. Biomat. Art. Cells & Immob. Biotech., 1991, 19, 675-686).
French Application Document No. 9,210,173 (1992) describes the use of a transacylation reaction between a polysaccharide ester, such as propylene glycol alginate (PGA), and a polyamine substance, such as a diamine or a protein, in order to manufacture microcapsules. The transacylation reaction between the ester and the polyamine is triggered in alkaline medium and produces a membrane formed of a polysaccharide associated with a polyamine by amide bonds. This document describes several processes for the preparation of microcapsules, all using a step of emulsification, during which either an aqueous phase is dispersed in a hydrophobic phase or a hydrophobic phase is dispersed in an aqueous phase. In all cases, the microcapsules become individualized from the initial emulsion, by basification of the emulsion.
If an attempt is made to apply this transacylation reaction directly to an aqueous suspension of spheres formed of a polysaccharide gelling in gelled form by a gelling agent such as a mono- or polyvalent cation, generation of a membrane around the spheres is not achieved. Thus, for example, if spheres are prepared by dropwise addition of a sodium alginate solution to an aqueous calcium solution, if the said spheres are then dispersed in an aqueous solution containing an esterified polysaccharide, such as propylene glycol alginate, and a protein, and if finally the aqueous suspension is basified so as to trigger the transacylation reaction between the esterified polysaccharide and the protein, the aqueous solution visibly sets in bulk as soon as the pH is sufficiently high to allow the reaction to take place.